Tornroth-Horsefield, S. | Search bioRxiv

bioRxiv2024

AER-270 and TGN-020 are not aquaporin-4 water channel blockers

Aquaporin-4 (AQP4) is the most abundant water channel protein in the brain. It controls water homeostasis, facilitates glymphatic function and is a drug target for brain edema following injury or stroke. Dysregulation of brain water homeostasis affects millions of people every year leading to death, disability and cognitive decline, for which no medicines are available. Two compounds, AER-270 and TGN-020, are sold as AQP4 inhibitors and a prodrug of AER-270 is currently in a phase I human trial. However, the direct effect of these compounds on AQP4 function has not been unequivocally demonstrated. Our data across multiple cellular and molecular assay systems demonstrate, unexpectedly, that AER-270 and TGN-020 do not inhibit AQP4. Although we observed an apparent inhibitory effect of AER-270 and TGN-020 in the Xenopus laevis oocyte assay, there was no effect in assays using reconstituted recombinant AQP4 or mammalian cells expressing exogenous or endogenous AQP4. We identify alternative mechanisms of action for both molecules that may explain previously reported in vivo results that were interpreted in the context of AQP4 inhibition. Overall, we conclude that AER-270 and TGN-020 should not be used to investigate the AQP4-dependence of biological processes in the brain.

biophysics↗

bioRxiv2022

Refining structural models of membrane proteins withdisordered domains in phospholipid nanodiscs

Small-angle scattering can be used to derive structural information about membrane proteins reconstituted in suitable carrier systems enabling solubilization of the membrane proteins in question. Since the studies are done in solution, there is no need for crystallization or deposition on sample grids, and it is in principle possible to obtain structural information about intrinsically disordered regions which cannot be resolved by crystallography or the quantitative link to which is hard to establish using e.g. electron microscopy methods. In this study, tetramers of the gated spinach aquaporin SoPIP2;1 were reconstituted into nanodiscs and small-angle x-ray scattering data were recorded. From these data, we refine structural models of the entire nanodisc-membrane protein complex including the flexible regions using newly developed models based on Fast Debye sums. We introduce software for these computations available via online repositories and discuss the implications and limitations of these methods. Author summaryWhen it comes to investigating the structure and function of the proteins, a particular class of proteins are known to be cumbersome and problematic: membrane proteins that reside in the cell membrane and regulate and facilitate a number of critical biological processes. Such proteins can often not be studied by conventional means as they unravel and denature structurally or even precipitate in solution. To add insult to injury, such membrane proteins also often contain parts that are intrinsically disordered rendering them irresolvable by e.g. traditional crystallographic techniques and hard to describe structurally. Here, we present a combined computational and experimental approach (as well as the necessary software) to analyze and determine the structure of such proteins in close-to-native conditions in so-called nanodiscs, a biological carrier systems, using small-angle scattering and molecular simulations.

biophysics↗